Nuclear matrix proteins

ABSTRACT

Nuclear matrix proteins (NMP) which are characterized by a defined expression in tissue are provided. These NMPs are useful markers in diagnosing and monitoring the stage of malignancy of a cell. Also provided are substantially purified polypeptides and nucleotide sequences encoding the NMPs of the invention.

This invention was made with Government support under NIH SPORE GrantP50 CA 58236-01 awarded by the National Institutes of Health, DK-19300National Institute of Arthritis, Diabetes and Digestive and KidneyDiseases and CA 15416, National Cancer Institute.

This application is a divisional of the application Ser. No. 08/015,624,filed on Feb. 9, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to nuclear matrix proteins andspecifically to novel nuclear matrix proteins with defined tissueexpression patterns in normal cells and cells associated with cellproliferative disorders.

2. Description of Related Art

Advances in recombinant DNA technology have led to the discovery ofnormal cellular genes (proto-oncogenes and tumor suppressor genes) whichcontrol growth, development, and differentiation. Under certaincircumstances, regulation-of these genes is altered and normal cellsassume neoplastic growth behavior. In some cases, the normal cellphenotype can be restored by various manipulations associated with thesegenes. There are over 40 known proto-oncogenes and suppressor genes todate, which fall into various categories depending on their functionalcharacteristics. These include, 1) growth factors and growth factorreceptors, 2) messengers of intracellular signal transduction pathways,for example, between the cytoplasm and the nucleus, and 3) regulatoryproteins influencing gene expression and DNA replication, located bothwithin and outside the nucleus.

During their life span, normal cells begin in an immature state withproliferative potential, pass through sequential stages ofdifferentiation, and eventually end in cell death. Cancer, on the otherhand, is a multistep process which can be defined in terms of stages ofmalignancy wherein the normal orderly progression is aberrant, probablydue to alterations in oncogenes, tumor suppressor genes, and othergenes. Research an oncogenes and their products has led to a morefundamental understanding of the mechanisms of cancer causation andmaintenance allowing more rational means of diagnosing and treatingmalignancies.

Genes associated with the control of normal growth and differentiationof cells include genes which encode regulatory proteins which influencegene expression and DNA replication. The gene products of many of thesegenes localize in the nucleus and many are DNA binding proteins. Thenucleus of an animal cell contains cellular DNA complexed with protein,referred to as chromatin. The chromatin is organized by the internalskeleton of the nucleus, called the nuclear matrix Nuclear matrixproteins (NMP) associated with DNA may be growth/differentiationregulatory proteins which play a role in the regulation of geneexpression in a cell. In cells that have lost their growth regulatorymechanisms, it can be envisioned that a nucleus-specific protein maycontinuously activate a transcriptional promoter region of a gene,causing over-expression of the gene. Similarly, a nuclear protein whichfunctions as a suppressor to control or suppress the expression ofvarious proto-oncogenes, may be under-expressed or expressed in a mutantform, thereby allowing aberrant expression of a gene which otherwisewould be suppressed.

Current cancer tests are generally nonspecific, insensitive and,consequently, of limited clinical application. For example, abiochemical test, widely used for both diagnostic and monitoring ofcancer, measures levels of carcinoembryonic antigen (CEA). CEA is anoncofetal antigen detectable in large amounts in embryonal tissue, butin small amounts in normal adult tissues. Serum of patients with certaingastrointestinal cancers contains elevated CEA levels that can bemeasured by immunological methods. The amount of CEA in serum correlateswith the remission or relapse of these tumors, with the levelsdecreasing abruptly after surgical removal of the tumor. The return ofelevated CEA levels signifies a return of malignant cells. CEA, however,is also a normal glycoprotein found at low levels in nearly all adults.Moreover, this protein can be elevated with several nonmalignantconditions and is not elevated in the presence of many cancers.Therefore it is far from ideal as a cancer marker. A similar oncofetaltumor marker is alpha-fetoprotein, an embryonic form of albumin. Again,the antigen is detectable in high amounts in embryonal tissue and in lowamounts in normal adults. It is elevated in a number of gastrointestinalmalignancies including hepatoma. Like CEA, a decrease correlates withthe remission of cancer and a re-elevation with relapse. There isinsufficient sensitive and specificity to make this marker useful forscreening for malignancy or for monitoring previously diagnosed cancerin any but a few selected cases.

In view of the foregoing, there remains a need for new cancer markerswhich would allow more effective diagnosis, prognosis and treatmentregimes. The identification of NMPs which are associated with theregulation of gene expression or cellular structure in normal and cancercells would provide ideal markers for identification of the stage ofmalignancy of a cell.

SUMMARY OF THE INVENTION

The present invention is based in the discovery of novel nuclear matrixproteins (NMP) which have defined patterns of tissue expression indifferent stages of abnormal growth and malignancy of a cell. The NMPsof the invention are defined by a tissue expression patterncharacteristic of any one of the proteins, NPB-1, NPB-2, NPB-3, NPB-4,NPB-5, NPB-6, NPB-7, NP-1, NP-2, NP-3, BPC-1, BPC-2, BPC-3, and PC-1.These proteins were initially identified as being associated with normalprostate tissue (NP), both normal and benign hyperplasia prostate tissue(NBP), benign hyperplasia and cancerous prostate tissue (BPC), orprostate cancer tissue (PC).

The invention provides nucleotides which encode the novel NMPs. The NMPsof the invention provide the basis for a method of detecting a cellproliferative disorder in a subject comprising contacting a cellularcomponent with a reagent which binds to the NMP. The method isespecially useful for detecting a cell proliferative disorder in atissue of the urogenital system, and specifically the prostate.

The invention also provides a method of treating a cell proliferativedisorder associated with NPB-1, NPB-2, NPB-3, NPB-4, NPB-5, NPB-6,NPB-7, NP-1, NP-2, NP-3, BPC-1, BPC-2, BPC-3, and PC-1. Such a methodmay include gene therapy, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C shows nuclear matrix protein composition of normal humanprostate (1A), benign prostatic hyperplasia (1B) and prostate cancer(1C). Large arrow panel A. variable group of proteins that wereinconsistently present on various types of tissue. Small arrows,proteins that are consistently changed in all tissues and are identifiedby molecular weight and isoelectric point in table 1. Abbreviations:LA--lamin A, LB--lamin B, LC--lamin C, A--actin, NP--normal prostate,NPB--normal prostate and BPH, BPC--BPH and prostate cancer, PC-prostatecancer, kD--molecular weight in thousands, SDS-PAGE--sodium dodecylsulfate-polyacrylamide gel electrophoresis and pl--isoelectric point

FIG. 2 shows specific nuclear matrix proteins in BPH and prostatecancer. Schematic of major tissue specific nuclear matrix proteins ofnormal prostate, BPH and prostate cancer. Abbreviations: kD--molecularweight in thousands, SDS-PAGE--sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and pl--isoelectric point and BPH--benign prostatichyperplasia.

FIG. 3 shows two models of multistep progression from normal prostate(Normal) to benign prostatic hyperplasia (BPH) or to prostate cancer(Cancer). Model I predicts that similar events occur in both pathways.Model II predicts different events occurring when progressing fromnormal to BPH as when progressing to cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides substantially pure nuclear matrixproteins (NMP) or functional fragments thereof, wherein the protein hasa tissue expression pattern characteristic of a protein selected fromthe group comprising NPB-1, NPB-2, NPB-3, NPB-4, NPB-5, NPB-6, NPB-7,NP-1, NP-2, NP-3, BPC-1, BPC-2, BPC-3, and PC-1. The invention alsoprovides polynucleotide sequences which encode these proteins. The NMPsof the invention can generally be characterized by their presence in acell during a specific stage of a cell proliferative disorder.

The term "cell-proliferative disorder" denotes malignant as well asnon-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignancy(i.e, cancer) is a multistep process and the proteins of the inventionare associated with three broad steps in the transition from a normalcell to a cancer cell. In broad stages, normal tissue (stage 1) maybegin to show signs of hyperplasia (stage 2) or show signs of neoplasia(stage 3). As used herein, "hyperplasia" refers to cells which exhibitabnormal multiplication or abnormal arrangement in a tissue. Included inthe term hyperplasia, are benign cellular proliferative disorders,including benign tumors. Proteins of the invention which exhibit atissue expression pattern in both normal tissue and benign hyperplastictissue (NPB) (non-malignant) include NPB-1, NPB-2, NPB-3, NPB-4, NPB-5,NPB-6 and NPB-7. The term "tissue expression pattern" refers to thesynthesis of a gene product of an NMP gene at a level which isdetectable by methods commonly used by those of skill in the art (e.g.,SDS-polyacrylamide gel electrophoresis). As used herein, "neoplasia"refers to abnormal new growth, which results in a tumor. Unlikehyperplasia, neoplastic proliferation persists even in the absence ofthe original stimulus and characterized as uncontrolled and progressive.Malignant neoplasms, or malignant tumors, are distinguished from benigntumors in that the former show a greater degree of anaplasia and havethe properties of invasion and metastasis. A protein of the invention,PC-1, is an example of a protein which has an expression pattern seen inmalignant neoplasms. The proteins of the invention, BPC-1, BPC-2 andBPC-3 are examples of proteins which are expressed in both benignhyperplastic tissue or tumors and also neoplastic tissues or tumors. Onthe other hand, proteins NP-1, NP-2 and NP-3 have tissue expressionpatterns in normal (non-tumor), non-hyperplastic, and non-neoplastictissue. Thus, the presence of these latter proteins essentially rule outthe presence of a cell proliferative disorder.

In summary, BPC-1, BPC-2 and BPC-3 and PC-1 are associated with tumorcells; NPB-1, NPB-2, NPB-3, NPB-4, NPB-5, NPB-6, and NPB-7 areassociated with normal and non-malignant hyperplastic or tumor cells;Pc-1 is associated with malignant cells; and NP-1, NP-2, and NP-3 areassociated with non-tumor cells. The term "associated with" refers tothe correlation between the expression pattern of the protein and thestage of progression to cancer.

The invention provides substantially pure NMPs or functional fragmentsthereof. As used herein, the term "functional polypeptide" or"functional fragment" refers to a polypeptide which possesses abiological function or activity which is identified through a definedfunctional assay and which is associated with a particular biologic,morphologic or phenotypic alteration in the cell. The polypeptidefragment possessing biological function can vary from as small as anepitope to which an antibody molecule can bind to as large as apolypeptide capable of participating in the characteristic induction orprogramming of phenotypic changes within a cell. It is understood thatall functional polypeptides encoding all or a functional portion of theNMPs of the invention are also included herein, so long as they arefound in the nuclear matrix and exhibit the tissue expression patterncharacteristic of a given NMP of the invention. A "functionalpolynucleotide" denotes a polynucleotide which encodes a functionalpolypeptide as described herein.

The term "substantially pure" or "isolated" means any NMP functionalpolypeptide of the present invention, or any gene encoding an NMPfunctional polypeptide, which is essentially free of other polypeptidesor genes, respectively, or of other contaminants with which it mightnormally be found in nature, and as such exists in a form not found innature. The invention also provides functional derivatives of the NMPsof the invention. By "functional derivative" is meant the "fragments,""variants," "analogues," or "chemical derivatives" of a molecule. A"fragment" of a molecule, such as any of the DNA or amino acid sequencesof the present invention, includes any nucleotide or amino acid subsetof the molecule. A "variant" of such molecule refers to a naturallyoccurring molecule substantially similar to either the entire molecule,or a fragment thereof. An "analog" of a molecule refers to a non-naturalmolecule substantially similar to either the entire molecule or afragment thereof.

Procedures which may be used to isolate the NMPs of the inventioninclude those commonly used for the separation of protein substancesincluding, for example; treatment of a sample containing NMP with commonprecipitants for proteins, followed by fractionation techniques such asion exchange chromatography, affinity chromatography, molecular sievechromatography, adsorption chromatography, ultrafiltration and variouscombinations thereof. The NMPs can be purified from a cell suspension bymethods described in U.S. Pat. Nos. 4,885,236 and 4,882,268, forexample. Other methods for purification of the polypeptides of theinvention will be known to those of skill in the art (see, for example,Current Protocols In Immunology, Coligan, et al., eds. 1992,incorporated herein by reference).

The NMP containing fractions can be subjected to SDS PAGE under suitableconditions and the gel slice containing NMP activity or corresponding tothe molecular weight of the NMP of interest is recovered. SDS PAGE isperformed according to the method of Laemmi, et al., (Nature, 227:680,1970) and is a technique well known to those in the art. Variations inconditions which are within a suitable range are understood to beencompassed within the purification procedure.

The NMP containing fraction from the SDS PAGE is subjected to reversephase HPLC and eluted with acetonitrile for example. The NMP which isobtained is substantially pure to permit N-terminal amino acidsequencing. The solution is dried under vacuum and redissolved in asmall volume of acetonitrile 95% +TFA (0.08%). The concentrated sampleis then introduced in a sequencer connected to a phenylthiohydantoine(PTH) analyzer.

The invention provides polynucleotides, such as DNA, cDNA, and RNA,encoding novel NMP functional polypeptides. It is understood that allpolynucleotides encoding all or a functional portion of the NMPs of theinvention are also included herein, so long as they are found in thenuclear matrix and exhibit the tissue expression pattern characteristicof a given NMP of the invention. Such polynucleotides include bothnaturally occurring and intentionally manipulated, for example,mutagenized polynucleotides.

The polynucleotide sequence-for NMP also includes antisense sequencesand sequences that are degenerate as a result of the genetic code. Thereare 20 natural amino acids, most of which are specified by more than onecodon. Therefore, as long as the amino acid sequence of NMP results in afunctional polypeptide (at least, in the case of the sensepolynucleotide strand), all degenerate nucleotide sequences are includedin the invention. Where the antisense polynucleotide is concerned, theinvention embraces all antisense polynucleotides capable of inhibitingproduction of the NMP polypeptide.

DNA sequences of the invention can be obtained by several methods. Forexample, the DNA can be isolated using hybridization procedures whichare well known in the art. These include, but are not limited to: 1)hybridization of probes to genomic or cDNA libraries to detect sharednucleotide sequences; 2) antibody screening of expression libraries todetect shared structural features; and 3) synthesis by the polymerasechain reaction (PCR). RNA sequences of the invention can be obtained bymethods known in the art (See for example, Current Protocols inMolecular Biology, Ausubel, et al. eds., 1989, incorporated herein byreference).

The development of specific DNA sequences encoding NMPs of the inventioncan be obtained by: (1) isolation of a double-stranded DNA sequence fromthe genomic DNA; (2) chemical manufacture of a DNA sequence to providethe necessary codons for the polypeptide of interest; and (3) in vitrosynthesis of a double-stranded DNA sequence by reverse transcription ofmRNA isolated from a eukaryotic donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA. Of these three methods for developingspecific DNA sequences for use in recombinant procedures, the isolationof genomic DNA isolates is the least common. This is especially truewhen it is desirable to obtain the microbial expression of mammalianpolypeptides due to the presence of introns.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired polypeptideproduct is known. When the entire sequence of amino acid residues of thedesired polypeptide is not known, the direct synthesis of DNA sequencesis not possible and the method of choice is the formation of cDNAsequences.

Among the standard procedures for isolating cDNA sequences of interestis the formation of plasmid carrying cDNA libraries which are derivedfrom reverse transcription of mRNA which is abundant in donor cells thathave a high level of genetic expression. When used in combination withpolymerase chain reaction technology, even rare expression products canbe cloned. In those cases where significant portions of the amino acidsequence of the polypeptide are known, the production of labeled singleor double-stranded DNA or RNA probe sequences duplicating a sequenceputatively present in the target cDNA may be employed in DNA/DNAhybridization procedures which are carried out on cloned copies of thecDNA which have been denatured into a single-stranded form (Jay, et al.,Nucleic Acid Research, 11:2325, 1983).

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample which includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. Hybridization is particularly useful inthe detection of cDNA clones derived from sources where an extremely lowamount of mRNA sequences relating to the polypeptide of interest arepresent. By using stringent hybridization conditions directed to avoidnon-specific binding, it is possible, for example, to allow theautoradiographic visualization of a specific cDNA clone by thehybridization of the target DNA to that single probe in the mixturewhich is its complete complement (Wallace, et al., Nucleic AcidResearch, 9:879, 1981).

An NMP-containing cDNA library can be screened by injecting the variouscDNAs into oocytes, allowing sufficient time for expression of the cDNAgene products to occur, and testing for the presence of the desired cDNAexpression product, for example, by using an antibody specific for NMPpolypeptide or by using functional assays for NMP activity and a tissueexpression pattern characteristic of NMP.

Screening procedures which rely on nucleic acid hybridization make itpossible to isolate any gene sequence from any organism, provided theappropriate probe is available. Oligonucleotide probes, which correspondto a part of the sequence encoding the protein in question, can besynthesized chemically. This requires that short, oligopeptide stretchesof amino acid sequence must be known. The DNA sequence encoding theprotein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixedaddition reaction which utilizes a heterogeneous mixture of denatureddouble-stranded DNA when the sequence is degenerate. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA.

Since the novel DNA sequences of the invention encode essentially all orpart of an NMP, it is now a routine matter to prepare, subclone, andexpress smaller polypeptide fragments of DNA from this or correspondingDNA sequences. Alternatively, by utilizing the DNA fragments disclosedherein which define the unique NMP classes of the invention it ispossible, in conjunction with known techniques, to determine the DNAsequences encoding the entire NMP. Such techniques are described in U.S.Pat. No. 4,394,443 and U.S. Pat. No. 4,446,235 which are incorporatedherein by reference.

A cDNA expression library, such as lambda gt11, can be screenedindirectly for NMP peptides having at least one epitope, usingantibodies specific for NMP. Such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of NMP cDNA.

Polyclonal antibodies are prepared by immunization of an animal, e.g.,rabbit, with an immunogenic sample of NMP followed by purification ofthe antibody by methods well known in the art

Antibodies provided in the present invention are immunoreactive with theNMP of the invention. Antibody which consists essentially of pooledmonoclonal antibodies with different epitopic specificities, as well asdistinct monoclonal antibody preparations are provided. Monoclonalantibodies are made from antigen containing fragments of the protein bymethods well known in the art (Kohler, et al., Nature, 256:495, 1975;Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).

The antibodies of the invention can be used in immunoaffinitychromatography for the isolation of sequences containing an NMP of thepresent invention. One way by which such immunoaffinity chromatographycan be utilized is by the binding of the antibodies of the invention toCNBr-Sepharose-4B or Tresyl activated Sepharose (Pharmacia). These solidphase-bound antibodies can then be used to specifically bind sequencescontaining NMP from mixtures of other proteins to enable isolation andpurification thereof. Bound NMP sequences can be eluted from theaffinity chromatographic material using techniques known to those ofordinary skill in the art such as, for example, chaotropic agents, lowpH, or urea

The production of an NMP DNA sequence can be accomplished byoligonucleotde(s) which are primers for amplification of the genomicpolynucleotide encoding an NMP. These unique oligonucleotde primers canbe produced based upon identification of the flanking regions contiguouswith the polynucleotide encoding the NMP. These oligonucleotide primerscomprise sequences which are capable of hybridizing with the flankingnucleotide sequence encoding an NMP polypeptide and sequencescomplementary thereto.

The primers of the invention include oligonucleotides of sufficientlength and appropriate sequence so as to provide specific initiation ofpolymerization on a significant number of nucleic acids in thepolynucleotide encoding the NMP. Specifically, the term "primer" as usedherein refers to a sequence comprising two or more deoxyribonucleotidesor ribonucleotides, preferably more than three, which sequence iscapable of initiating synthesis of a primer extension product, which issubstantially complementary to an NMP strand. Experimental conditionsconducive to synthesis include the presence of nucleoside triphosphatesand an agent for polymerization and extension, such as DNA polymeraseand a suitable temperature and pH. The primer is preferably singlestranded for maximum efficiency in amplification, but may be doublestranded. If double stranded, the primer is first treated to separatethe two strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent for polymerization and extension of thenucleotides. The exact length of primer will depend on many factors,including temperature, buffer, and nucleotide composition. Theoligonucleotide primer typically contains 15-22 or more nucleotides,although it may contain fewer nucleotides.

Primers of the invention are designed to be "substantially"complementary to each strand of polynucleotide encoding the NMP to beamplified. This means that the primers must be sufficientlycomplementary to hybridize with their respective strands underconditions which allow the agent for polymerization and nucleotideextension to act. In other words, the primers should have sufficientcomplementarity with the flanking sequences to hybridize therewith andpermit amplification of the polynucleotide encoding the receptor NMP.Preferably, the primers have exact complementarity with the flankingsequence strand.

Oligonucleotide primers of the invention are employed in theamplification process which is an enzymatic chain reaction that producesexponential quantities of polynucleotide encoding the NMP relative tothe number of reaction steps involved. Typically, one primer iscomplementary to the negative (-) strand of the polynucleotide encodingthe NMP and the other is complementary to the positive (+) strand.Annealing the primers to denatured nucleic acid followed by extensionwith an enzyme, such as the large fragment of DNA Polymerase I (Klenow)and nucleotides, results in newly synthesized (+) and (-) strandscontaining the NMP sequence. Because these newly synthesized sequencesare also templates, repeated cycles of denaturing, primer annealing, andextension results in exponential production of the sequence (i.e., theNMP polynucleotide sequence) defined by the primer. The product of thechain reaction is a discrete nucleic acid duplex with terminicorresponding to the ends of the specific primers employed. Those ofskill in the art will know of other amplification methodologies whichcan also be utilized to increase the copy number of target nucleic acid.These may include, for example, ligation activated transcription (LAT),ligase chain reaction (LCR), and strand displacement activation (SDA),although PCR is the preferred method.

The oligonucleotide primers of the invention may be prepared using anysuitable method, such as conventional phosphotriester and phosphodiestermethods or automated embodiments thereof. In one such automatedembodiment, diethylphosphoramidites are used as starting materials andmay be synthesized as described by Beaucage, et al. (TetrahedronLetters, 2:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. One method of amplification which can be used accordingto this invention is the polymerase chain reaction (PCR) described inU.S. Pat. Nos. 4,683,202 and 4,683,195.

Any nucleic acid specimen, in purified or nonpurified form, can beutilized as the starting nucleic acid for the above procedures, providedit contains, or is suspected of containing, the specific nucleic acidsequence of an NMP of the invention. Thus, the process may employ, forexample, DNA or RNA (including mRNA), wherein DNA or RNA may be singlestranded or double stranded. In the event that RNA is to be used as atemplate, enzymes, and/or conditions optimal for reverse transcribingthe template to DNA would be utilized. In addition, a DNA-RNA hybridwhich contains one strand of each may be utilized. A mixture of nucleicacids may also be employed, or the nucleic acids produced in a previousamplification reaction herein, using the same or different primers maybe so utilized. The specific nucleic acid sequence to be amplified,(i.e., NMP sequence), may be a fraction of a larger molecule or can bepresent initially as a discrete molecule, so that the specific sequenceconstitutes the entire nucleic acid. It is not necessary that thesequence to be amplified be present initially in a pure form; it may bea minor fraction of a complex mixture, such as contained in whole humanDNA.

DNA or RNA utilized herein may be extracted from a body sample, such asprostate tissue, or various other tissue, by a variety of techniquessuch as that described by Maniatis, et al. (Molecular Cloning, 280:281,1982). If the extracted sample is impure (such as plasma, serum,ejaculate or blood), it may be treated before amplification with anamount of a reagent effective to open the cells, fluids, tissues, oranimal cell membranes of the sample, and to expose and/or separate thestrand(s) of the nucleic acid(s). This lysing and nucleic aciddenaturing step to expose and separate the strands will allowamplification to occur much more readily.

Where the target nucleic acid sequence of the sample contains twostrands, it is necessary to separate the strands of the nucleic acidbefore it can be used as the template. Strand separation can be effectedeither as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing various suitable denaturing conditions, including physical,chemical, or enzymatic means, the word "denaturing" includes all suchmeans. One physical method of separating nucleic acid strands involvesheating the nucleic acid until it is denatured. Typical heatdenaturation may involve temperatures ranging from about 80° to 105° C.for times ranging from about 1 to 10 minutes. Strand separation may alsobe induced by an enzyme from the class of enzymes known as helicases orby the enzyme RecA, which has helicase activity, and in the presence ofriboATP, is known to denature DNA. The reaction conditions suitable forstand separation of nucleic acids with helicases are described by KuhnHoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and techniquesfor using RecA are reviewed in C. Radding (Ann. Rev. Genetics,16:405-437, 1982).

If the nucleic acid containing the sequence to be amplified is singlestranded, its complement is synthesized by adding one or twooligonucleotide primers. If a single primer is utilized, a primerextension product is synthesized in the presence of primer, an agent forpolymerization, and the four nucleoside triphosphates described below.The product will be partially complementary to the single-strandednucleic acid and will hybridize with a single-stranded nucleic acid toform a duplex of unequal length strands that may then be separated intosingle strands to produce two single separated complementary strands.Alternatively, two primers may be added to the single-stranded nucleicacid and the reaction carried out as described.

When complementary strands of nucleic acid or acids are separated,regardless of whether the nucleic acid was originally double or singlestranded, the separated strands are ready to be used as a template forthe synthesis of additional nucleic acid strands. This synthesis isperformed under conditions allowing hybridization of primers totemplates to occur. Generally synthesis occurs in a buffered aqueoussolution, preferably at a pH of 7-9, most preferably about 8.Preferably, a molar excess (for genomic nucleic acid, usually about 10⁸:1 primer:template) of the two oligonucleotide primers is added to thebuffer containing the separated template strands. It is understood,however, that the amount of complementary strand may not be known if theprocess of the invention is used for diagnostic applications, so thatthe amount of primer relative to the amount of complementary strandcannot be determined with certainty. As a practical matter, however, theamount of primer added will generally be in molar excess over the amountof complementary strand (template) when the sequence to be amplified iscontained in a mixture of complicated long-chain nucleic acid strands. Alarge molar excess is preferred to improve the efficiency of theprocess.

The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP areadded to the synthesis mixture, either separately or together with theprimers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period, the solution is allowed to cool toroom temperature, which is preferable for the primer hybridization. Tothe cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein "agent for polymerization"),and the reaction is allowed to occur under conditions known in the art.The agent for polymerization may also be added together with the otherreagents if it is heat stable. This synthesis (or amplification)reaction may occur at room temperature up to a temperature above whichthe agent for polymerization no longer functions. Thus, for example, ifDNA polymerase is used as the agent, the temperature is generally nogreater than about 40° C. Most conveniently the reaction occurs at roomtemperature.

The agent for polymerization may be any compound or system which willfunction to accomplish the synthesis of primer extension products,including enzymes. Suitable enzymes for this purpose include, forexample, E. coil DNA polymerase I, Klenow fragment of E. coli DNApolymerase I, T4 DNA polymerase, other available DNA polymerases,polymerase muteins, reverse transcriptase, and other enzymes, includingheat-stable enzymes (i.e., those enzymes which perform primer extensionafter being subjected to temperatures sufficiently elevated to causedenaturation). Suitable enzymes will facilitate combination of thenucleotides in the proper manner to form the primer extension productswhich are complementary to each NMP nucleic acid strand. Generally, thesynthesis will be initiated at the 3' end of each primer and proceed inthe 5' direction along the template strand, until synthesis terminates,producing molecules of different lengths. There may be agents forpolymerization, however, which initiate synthesis at the 5' end andproceed in the other direction, using the same process as describedabove.

The newly synthesized NMP strand and its complementary nucleic acidstrand will form a double-stranded molecule under hybridizing conditionsdescribed above and this hybrid is used in subsequent steps of theprocess. In the next step, the newly synthesized double-strandedmolecule is subjected to denaturing conditions using any of theprocedures described above to provide single-stranded molecules.

The above process is repeated on the single-stranded molecules.Additional agent for polymerization, nucleotides, and primers may beadded, if necessary, for the reaction to proceed under the conditionsprescribed above. Again, the synthesis will be initiated at one end ofeach of the oligonucleotide primers and will proceed along the singlestrands of the template to produce additional nucleic acid. After thisstep, half of the extension product will consist of the specific nucleicacid sequence bounded by the two primers.

The steps of denaturing and extension product synthesis can be repeatedas often as needed to amplify the NMP nucleic acid sequence to theextent necessary for detection. The amount of the specific nucleic acidsequence produced will accumulate in an exponential fashion.

Sequences amplified by the methods of the invention can be furtherevaluated, detected, cloned, sequenced, and the like, either in solutionor after binding to a solid support, by any method usually applied tothe detection of a specific DNA sequence such as PCR, oligomerrestriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985),allele-specific oligonucleotide (ASO) probe analysis (Conner, et al.,Proc. Nail. Acad. Sci. USA, 80:278, 1983), oligonucleotde ligationassays (OLAs) (Landegren, et al., Science, 241:1077, 1988), and thelike. Molecular techniques for DNA analysis have been reviewed(Landegren, et al., Science, 242:229-237, 1988).

DNA sequences encoding NMP can be expressed in vitro by DNA transferinto a suitable host cell. "Host cells" are cells in which a vector canbe propagated and its DNA expressed. The term also includes any progenyof the subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term"host cell" is used. Methods of stable transfer, in other words when theforeign DNA is continuously maintained in the host, are known in theart.

In the present invention, the NMP polynucleotide sequences may beinserted into a recombinant expression vector. The term "recombinantexpression vector" refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theNMP nucleic acid sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertednucleic acid sequence of the host. The expression vector typicallycontains an origin of replication, a promoter, as well as specific geneswhich allow phenotypic selection of the transformed cells. Vectorssuitable for use in the present invention include, but are not limitedto the T7-based expression vector for expression in bacteria (Rosenberget al., Gene 56:125, 1987), the pMSXND expression vector for expressionin mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein 1, orpolyhedrin promoters).

Polynucleotide sequences encoding NMP can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method byprocedures well known in the art. Alternatively, MgCl₆ or RbCl can beused. Transformation can also be performed after forming a protoplast ofthe host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the NMP of the invention, anda second foreign DNA molecule encoding a selectable phenotype, such asthe herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982).

Isolation and purification of microbially expressed protein, orfragments thereof provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.Antibodies provided in the present invention are immunoreactive with NMPpolypeptide or fragments thereof.

The NMP polypeptide of the invention includes fragments and conservativevariations of the polypeptides. Minor modifications of the NMP primaryamino acid sequence may result in proteins which have substantiallyequivalent activity as compared to the NMP polypeptide described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polypeptides produced by thesemodifications are included herein as long as the biological activity ofNMP still exists. Further, deletion of one or more amino acids can alsoresult in a modification of the structure of the resultant moleculewithout significantly altering its biological activity. This can lead tothe-development of a smaller active molecule which would have broaderutility.

The term "conservative variation" as used herein denotes the replacementof an amino acid residue by another, biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like.

Peptides of the invention can be synthesized by the well known solidphase peptide synthesis methods described Merrifield, J. Am. Chem. Sac.,85:2149, 1962), and Stewart and Young, Solid Phase Peptides Synthesis,(Freeman, San Francisco, 1969, pp27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout 1/4-1 hours at 0° C. After evaporation of the reagents, thepeptides are extracted from the polymer with 1% acetic acid solutionwhich is then lyophilized to yield the crude material. This can normallybe purified by such techniques as gel filtration on Sephadex G-15 using5% acetic acid as a solvent. Lyophilization of appropriate fractions ofthe column will yield the homogeneous peptide or peptide derivatives,which can then be characterized by such standard techniques as aminoacid analysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

The invention includes polyclonal and monoclonal antibodiesimmunoreactive with NMP polypeptide or immunogenic fragments thereof. Ifdesired, polygonal antibodies can be further purified, for example, bybinding to and elution from a matrix to which NMP polypeptide is bound.Those of skill in the art will know of various other techniques commonin the immunology arts for purification and/or concentration ofpolyclonal antibodies, as well as monoclonal antibodies. Antibody whichconsists essentially of pooled monoclonal antibodies with differentepitopic specificities, as well as distinct monoclonal antibodypreparations are provided. The term antibody or, immunoglobulin as usedin this invention includes intact molecules as well as fragmentsthereof, such as Fab and F(ab')₂, which are functionally capable ofbinding an epitopic determinant on NMP.

A preferred method for the identification and isolation of antibodybinding domain which exhibit binding with NMP is the bacteriophage λvector system. This vector system has been used to express acombinatorial library of Fab fragments from the mouse antibodyrepertoire in Escherichia coil (Huse, et al., Science, 246:1275-1281,1989) and from the human antibody repertoire (Mullinax, et al., Proc.Natl. Acad. Sci., 87:8095-8099, 1990). As described therein, receptors(Fab molecules) exhibiting binding for a preselected ligand wereidentified and isolated from these antibody expression libraries. Thismethodology can also be applied to hybridoma cell lines expressingmonoclonal antibodies with binding for a preselected ligand. Hybridomaswhich secrete a desired monoclonal antibody can be produced in variousways using techniques well understood by those having ordinary skill inthe art and will not be repeated here. Details of these techniques aredescribed in such references as Monoclonal Antibodies-Hybridomas: A NewDimension in Biological Analysis, Edited by Roger H. Kennett et al.,Plenum Press, 1980; and U.S. Pat. No. 4,172,124, incorporated herein byreference.

As used herein, the term "cell-proliferative disorder" denotes malignantas well as non-malignant (or benign) disorders. This term furtherencompasses hyperplastic disorders. The cells comprising theseproliferative disorders often appear morphologically and genotypicallyto differ from the surrounding normal tissue. As noted above,cell-proliferative disorders may be associated, for example, withexpression or absence of expression of the NMPs of the invention.Expression of NMP at an inappropriate time during the cell cycle or inan incorrect cell type may result in a cell-proliferative disorder. TheNMP polynucleotide in the form of an antisense polynucleotide is usefulin treating hyperplasia and malignancies of the various organ systems,particularly, for example, those of urogenital origin such as theprostate. In addition, hyperplasia and malignancies of such organs asthe kidney and bladder can be treated using the NMP polynucleotides ofthe invention. Essentially, any disorder which is etiologically linkedto expression of NMP could be considered susceptible to treatment with areagent of the invention which modulates NMP expression. The term"modulate" envisions the suppression of expression of NMP when it isinappropriately expressed or augmentation of NMP expression when it isunder-expressed or when the NMP expressed is a mutant form of thepolypeptide. When a cell-proliferative disorder is associated with NMPexpression, (e.g., BPC-1, 2, 3 and PC-1), such suppressive reagents asantisense NMP polynucleotide sequence or NMP binding antibody can beintroduced to a cell. Alternatively, when a cell proliferative disorderis associated with under-expression or expression of a mutant NMPpolypeptide (e.g., NPB 1-7 and NP-1-3), a sense polynucleotide sequence(the DNA coding strand) or NMP polypeptide can be introduced into thecell.

The invention provides a method for detecting a cell expressing NMP or acell proliferative disorder associated with NMP in a subject comprisingcontacting a cell component suspected of expressing NMP or having a NMPassociated disorder, with a reagent which binds to the component. Thecell component can be nucleic acid, such as DNA or RNA, or protein. Whenthe component is nucleic acid, the reagent is a nucleic acid probe orPCR primer. When the cell component is protein, the reagent is anantibody probe. The probes are directly or indirectly detectablylabeled, for example, with a radioisotope, a fluorescent compound, abioluminescent compound, a chemiluminescent compound, a metal chelator,or an enzyme. Those of ordinary skill in the art will know of othersuitable labels for binding to the probe or will be able to ascertainsuch, using routine experimentation.

For purposes of the invention, an antibody or nucleic acid probespecific for NMP may be used to detect the presence of NMP polypeptide(using antibody) or polynucleotide (using nucleic acid probe) inbiological fluids or tissues. Oligonucleotide primers based on anycoding sequence region in the NMP sequence are useful for amplifyingDNA, for example by PCR. Any specimen containing a detectable amount ofantigen can be used. A preferred sample of this invention, especiallyfor detecting prostate cancer, is tissue of urogenital origin,specifically tissue of the prostate. Alternatively, biological fluidswhich may contain cells indicative of an NMP-associatedcell-proliferative disorder, such as ejaculate or urine, may be used.Preferably the subject is human.

Another technique which may also result in greater sensitivity consistsof coupling the probe to low molecular weight haptens. These haptens canthen be specifically detected by means of a second reaction. Forexample, it is common to use such haptens as biotin, which reacts withavidin, or dinitrophenol, pyridoxal, and fluorescein, which can reactwith specific antihapten antibodies.

The method for detecting a cell expressing a particular NMP of theinvention or a cell-proliferative disorder associated with an NMP,described above, can be utilized for detection of residual prostatecancer or other malignancies or benign hyperplasia conditions in asubject in a state of clinical remission. Additionally, the method fordetecting NMP polypeptide in cells is useful for detecting acell-proliferative disorder by identifying cells expressing specificNMPs in comparison with NMPs expressed in normal cells. Using the methodof the invention, NMP expression can be identified in a cell and theappropriate course of treatment can be employed (e.g., sense orantisense gene therapy, as well as conventional chemotherapy). Since theexpression pattern of the NMPs of the invention vary with the stage ofmalignancy of a cell, a sample such as prostate issue can be screenedwith a panel of NMP-specific reagents (e.g., nucleic acid probes orantibodies to NMPs) to detect NMP expression and diagnose the stage ofmalignancy of the cell.

The monoclonal antibodies of the invention are suited for use, forexample, in immunoassays in which they can be utilized in liquid phaseor bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays which can utilize monoclonalantibodies of the invention are competitive and noncompetitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays which arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Alternatively,antibody of the invention can be used to detect NMPs present inelectrophoretically dispersed gel protocols such as Western blots and2-dimensional gels. Those of skill in the art will know, or can readilydiscern, other immunoassay formats without undue experimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of NMP. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such using routineexperimentation.

In performing the assays it may be desirable to include certain"blockers" in the incubation medium (usually added with the labeledsoluble antibody). The "blockers" are added to assure that non-specificproteins, proteases, or anti-heterophilic immunoglobulins to anti-NMPimmunoglobulins present in the experimental sample do not cross-ink ordestroy the antibodies on the solid phase support, or the radiolabeledindicator antibody, to yield false positive or false negative results.The selection of "blockers" therefore may add substantially to thespecificity of the assays described in the present invention.

It has been found that a number of nonrelevant (i.e., nonspecific)antibodies of the same class or subclass (isotype) as those used in theassays (e.g., IgG1, IgG2a, IgM, etc.) can be used as "blockers". Theconcentration of the "blockers" (normally 1-100 μg/μl) is important, inorder to maintain the proper sensitivity yet inhibit any unwantedinterference by mutually occurring cross reactive proteins in thespecimen.

As used in this invention, the term "epitope" includes any determinantcapable of specific interaction with the monoclonal antibodies of theinvention. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics.

In using the monoclonal antibodies of the invention for the in vivodetection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term"diagnostically effective" means that the amount of detectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the NMP antigen for which the monoclonalantibody is specific.

The concentration of detectably labeled monoclonal antibody which isadministered should be sufficient such that the binding to those cellshaving NMP is detectable compared to the background. Further, it isdesirable that the detectably labeled monoclonal antibody be rapidlycleared from the circulatory system in order to give the besttarget-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for invivo diagnosis will vary depending on such factors as age, sex, andextent of disease of the individual. The dosage of monoclonal antibodycan vary from about 0.001 mg/m² to about 500 mg/m², preferably 0.1 mg/m²to about 200 mg/m², most preferably about 0.1 mg/m² to about 10 mg/m².Such dosages may vary, for example, depending on whether multipleinjections are given, tumor burden, and other factors known to those ofskill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to immunoglobulineither directly or indirectly by using an intermediate functional group.Intermediate functional groups which often are used to bindradioisotopes which exist as metallic ions to immunoglobulins are thebifunctional chelating agents such as diethylenetriaminepentacetic acid(DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Typical examples of metallic ions which can be bound to the monoclonalantibodies of the invention are ¹¹¹ In, ⁹⁷ Ru, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹Zr, and ²⁰¹ TI.

The monoclonal antibodies of the invention can also be labeled with aparamagnetic isotope for purposes of in vivo diagnosis, as in magneticresonance imaging (MRI) or electron spin resonance (ESR). In general,any conventional method for visualizing diagnostic imaging can beutilized. Usually gamma and positron emitting radioisotopes are used forcamera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵²Cr, and ⁵⁶ Fe.

The monoclonal antibodies of the invention can be used to monitor thecourse of amelioration of NMP associated cell-proliferative disorder.Thus, by measuring the increase or decrease in the number of callsexpressing a NMP or changes in NMP present in various body fluids, suchas ejaculate or urine, it would be possible to determine whether aparticular therapeutic regiment aimed at ameliorating the disorder iseffective.

The monoclonal antibodies of the invention can also be used, alone or incombination with effector cells (Douillard, et al. Hybridoma, 5Supp.1:S139, 1986), for immunotherapy in an animal having a cellproliferative disorder which expresses NMP polypeptide with epitopesreactive with the monoclonal antibodies of the invention.

When used for immunotherapy, the monoclonal antibodies of the inventionmay be unlabeled or labeled wfi a therapeutic agent. These agents can becoupled either directly or indirectly to the monoclonal antibodies ofthe invention. One example of indirect coupling is by use of a spacermoiety. These spacer moieties, in turn, can be either insoluble orsoluble (Diener, et al., Science, 231:148, 1986) and can be selected toenable drug release from the monoclonal antibody molecule at the targetsite. Examples of therapeutic agents which can be coupled to themonoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

The drugs which can be conjugated to the monoclonal antibodies of theinvention include non-proteinaceous as well as proteinaceous drugs. Theterms "non-proteinaceous drugs" encompasses compounds which areclassically referred to as drugs, for example, mitomycin C,daunorubicin, and vinblastine.

The proteinaceous drugs with which the monoclonal antibodies of theinvention can be labeled include immunomodulators and other biologicalresponse modifiers. The term "biological response modifiers" encompassessubstances which are involved in modifying the immune response in suchmanner as to enhance the destruction of an NMP-associated tumor forwhich the monoclonal antibodies of the invention are specific. Examplesof immune response modifiers include such compounds as lymphokines.Lymphokines include tumor necrosis factor, the interleukins,lymphotoxin, macrophage activating factor, migration inhibition factor,colony stimulating factor, and interferon. Interferons with which themonoclonal antibodies of the invention can be labeled includealpha-interferon, beta-interferon and gamma-interferon and theirsubtypes.

In using radioisotopically conjugated monoclonal antibodies of theinvention for immunotherapy certain isotypes may be more preferable thanothers depending on such factors as tumor cell distribution as well asisotope stability and emission. If desired, the tumor cell distributioncan be evaluated by the in vivo diagnostic techniques described above.Depending on the cell proliferative disease some emitters may bepreferable to others. In general, alpha and beta particle-emittingradioisotopes are preferred in immunotherapy.

For example, if an animal has solid tumor foci a high energy betaemitter capable of penetrating several millimeters of tissue, such as ⁹⁰Y, may be preferable. On the other hand, if the cell proliferativedisorder consists of simple target cells, as in the case of leukemia, ashort range, high energy alpha emitter, such as ²¹² Bi, may bepreferable. Examples of radioisotopes which can be bound to themonoclonal antibodies of the invention for therapeutic purposes are ¹²⁵I, ¹³¹ I, ⁹⁰ Y, ⁶⁷ Cu, ²¹² Bi, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, ⁶⁵ Zn, and¹⁸⁸ Re.

Lectins are proteins, usually isolated from plant material, which bindto specific sugar moieties. Many lectins are also able to agglutinatecells and stimulate lymphocytes. However, ricin is a toxic lectin whichhas been used immunotherapeutically. This is preferably accomplished bybinding the alpha-peptide chain of ricin, which is responsible fortoxicity, to the antibody molecule to enable site specific delivery ofthe toxic effect.

Toxins are poisonous substances produced by plants, animals, ormicroorganisms, that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A componentcan be bound to an antibody and used for site specific delivery to a NMPbearing cell. Other therapeutic agents which can be coupled to themonoclonal antibodies of the invention are known, or can be easilyascertained, by those of ordinary skill in the art.

The labeled or unlabeled monoclonal antibodies of the invention can alsobe used in combination with therapeutic agents such as those describedabove. Especially preferred are therapeutic combinations comprising themonoclonal antibody of the invention and immunomodulators and otherbiological response modifiers.

Thus, for example, the monoclonal antibodies of the invention can beused in combination with alpha-interferon. This treatment modalityenhances monoclonal antibody targeting of carcinomas by increasing theexpression of monoclonal antibody reactive antigen by the carcinomacells (Greiner, et al., Science, 235:895, 1987). Alternatively, themonoclonal antibody of the invention could be used, for example, incombination with gamma-interferon to thereby activate and increase theexpression of Fc receptors by effector cells which, in turn, results inan enhanced binding-of the monoclonal antibody to the effector cell andkilling of target tumor cells. Those of skill in the art will be able toselect from the various biological response modifiers to create adesired effector function which enhances the efficacy of the monoclonalantibody of the invention.

When the monoclonal antibody of the invention is used in combinationwith various therapeutic agents, such as those described herein, theadministration of the monoclonal antibody and the therapeutic agentusually occurs substantially contemporaneously. The term "substantiallycontemporaneously" means that the monoclonal antibody and thetherapeutic agent are administered reasonably close together withrespect to time. Usually, it is preferred to administer the therapeuticagent before the monoclonal antibody. For example, the therapeutic agentcan be administered 1 to 6 days before the monoclonal antibody. Theadministration of the therapeutic agent can be daily, or at any otherinterval, depending upon such factors, for example, as the nature of thetumor, the condition of the patient and half-life of the agent.

Using monoclonal antibodies of the invention, it is possible to designtherapies combining all of the characteristics described herein. Forexample, in a given situation it may be desirable to administer atherapeutic agent, or agents, prior to the administration of themonoclonal antibodies of the invention in combination with effectorcells and the same, or different, therapeutic agent or agents. Forexample, it may be desirable to treat patients with prostate, kidney orbladder carcinoma by first administering gamma-interferon andinterleukin-2 daily for 3 to 5 days, and on day 5 administer themonoclonal antibody of the invention in combination with effector cellsas well as gamma-interferon, and interleukin-2.

It is also possible to utilize liposomes with the monoclonal antibodiesof the invention in their membrane to specifically deliver the liposometo the tumor expressing NMP. These liposomes can be produced such thatthey contain, in addition to the monoclonal antibody, suchimmunotherapeutic agents as those described above which would then bereleased at the tumor site (Wolff, et al., Biochemical et BiophysicalActa, 802:259, 1984).

The dosage ranges for the administration of monoclonal antibodies of theinvention are those large enough to produce the desired effect in whichthe symptoms of the malignant disease are ameliorated. The dosage shouldnot be so large as to cause adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex and extent of the diseasein the patient and can be determined by one of skill in the art. Thedosage can be adjusted by the individual physician in the event of anycomplication. Dosage can vary from about 0.1 mg/kg to about 2000 mg/kg,preferably about 0.1 mg/kg to about 500 mg/kg, in one or more doseadministrations daily, for one or several days. Generally, when themonoclonal antibodies of the invention are administered conjugated withtherapeutic agents, lower dosages, comparable to those used for in vivodiagnostic imaging, can be used.

The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

The present invention also provides a method for treating a subject withan NMP-associated cell-proliferative disorder using an NMP nucleotidesequence. An NMP nucleotide sequence which may encode a suppressorpolypeptide may be under-expressed as compared to expression in a normalcell, therefore it is possible to design appropriate therapeutic ordiagnostic techniques directed to this sequence. Thus, where acell-proliferative disorder is associated with the expression of an NMPassociated with malignancy, nucleic acid sequences that interfere withNMP expression at the translational level can be used. This approachutilizes, for example, antisense nucleic acid and ribozymes to blocktranslation of a specific NMP mRNA, either by masking that mRNA with anantisense nucleic acid or by cleaving it with a ribozyme. In cases whena cell proliferative disorder or abnormal cell phenotype is associatedwith the under expression of NMP suppressor for example, nucleic acidsequences encoding NMP (sense) could be administered to the subject withthe disorder.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule (Weintraub, ScientificAmerican, 262:40, 1990). In the cell, the antisense nucleic acidshybridize to the corresponding mRNA, forming a double-stranded molecule.The antisense nucleic acids interfere with the translation of the mRNAsince the cell will not translate a mRNA that is double-stranded.Antisense oligomers of about 15 nucleotides are preferred, since theyare easily synthesized and are less likely to cause problems than largermolecules when introduced into the target NMP-producing cell. The use ofantisense methods to inhibit the in vitro translation of genes is wellknown in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single-stranded RNA in a manner analogous to DNArestriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J.Amer.Med Assn., 260:3030, 1988). A major advantage ofthis approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while "hammerhead"-type is ribozymes recognize base sequences11-18 bases in length. The longer the recognition sequence, the greaterthe likelihood that that sequence will occur exclusively in the targetmRNA species. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

The present invention also provides gene therapy for the treatment ofcell proliferative disorders which are mediated by NMP. Such therapywould achieve its therapeutic effect by introduction of the appropriateNMP polynucleotide (antisense or sense), into cells of subjects havingthe proliferative disorder. Delivery of antisense NMP polynucleotide canbe achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system. Disorders associated withunder-expression of an NMP or expression of a cancer-associated NMP,could be treated using gene therapy with sense or antisense nucleotidesequences, respectively.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a NMP sequence of interestinto the viral vector, along with another gene which encodes the ligandfor a receptor on a specific target cell, for example, the vector is nowtarget specific. Retroviral vectors can be made target specific byinserting, for example, a polynucleotide encoding a sugar, a glycolipid,or a protein. Preferred targeting is accomplished by using an antibodyto target the retroviral vector. Those of skill in the art will know of,or can readily ascertain without undue experimentation, specificpolynucleotide sequences which can be inserted into the retroviralgenome to allow target specific delivery of the retroviral vectorcontaining the NMP sense or antisense polynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsidation. Helper cell lines which havedeletions of the packaging signal include but are not limited to ψ2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for NMP antisense polynucleotides acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. It has beenshown that large unilamellar vesicles (LUV), which range in size from0.2-4.0 urn can encapsulate a substantial percentage of an aqueousbuffer containing large macromolecules. RNA, DNA and intact virions canbe encapsulated within the aqueous interior and be delivered to cells ina biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,1981). In addition to mammalian cells, liposomes have been used fordelivery of polynucleotides in plant, yeast and bacterial cells. Inorder for a liposome to be an efficient gene transfer vehicle, thefollowing characteristics should be present: (1) encapsulation of thegenes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes has been classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In general, the compounds bound to the surface of the targeted deliverysystem will be ligands and receptors which will allow the targeteddelivery system to find and "home in" on the desired cells. A ligand maybe any compound of interest which will bind to another compound, such asa receptor.

In general, surface membrane proteins which bind to specific effectormolecules are referred to as receptors. In the present invention,antibodies of the invention are preferred receptors. Antibodies can beused to target liposomes to specific cell-surface ligands, in this casethe NMPs of choice. Preferably, the target tissue is urogenital andspecifically is prostate tissue. Kidney and bladder tissue may also beutilized. A number of procedures can be used to covalently attach eitherpolyclonal or monoclonal antibodies to a liposome bilayer.Antibody-targeted liposomes can include monoclonal or polyclonalantibodies or fragments thereof such as Fab, or F(ab')₂, as long as theybind efficiently to an the antgenic epitope on the target cells.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the polynucleotides or themonoclonal antibodies of the invention, the medicament being used fortherapy of NMP associated cell proliferative disorders.

The NMPs of the invention are useful as a screening tool forcompositions which affect an NMP of a cell. Thus, in another embodiment,the invention provides a method for identifying a composition whichaffects an NMP comprising incubating the components, which include thecomposition to be tested and the cell (or cell suspension), underconditions sufficient to allow the components to interact, thensubsequently measuring the effect of the composition on the NMP. Theobserved effect on the NMP may be either inhibitory or stimulatory.

For example, in a malignant cell of the prostate, compositions which areinhibitory to BPC-1, BPC-2, BPC-3, or PC-1 expression can be identifiedby measuring the level of the NMP in the cell or cell extract before andafter treatment with the composition. Alternatively, the levels of NP-1,NP-2, or NP-3 can be monitored to identify compositions which stimulateexpression of these NMPs, found in normal cells.

The material for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method.

For example, one of the container means may comprise a probe which is orcan be detectably labelled. Such probe may be an antibody or nucleotidespecific for a target protein or a target nucleic acid, respectively,wherein the target is indicative, or correlates with, the presence of anNMP of the invention. Where the kit utilizes nucleic acid hybridizationto detect the target nucleic acid, the kit may also have containerscontaining nucleotide(s) for amplification of the target nucleic acidsequence and/or a container comprising a reporter-means, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic, florescent, or radionuclidelabel.

The following Examples are intended to illustrate, but not to limit theinvention. While such Examples are typical of those that might be used,other procedures known to those skilled in the art may alternatively beutilized.

EXAMPLE 1 Identification and Purification of Nuclear Matrix Proteins

Patients

Fresh prostatic tissue was studied from 21 men undergoing radicalretropubic prostatectomy for clinically localized (Stage B, T2) prostatecancer (N-19) (Gleason grade 5-9) or open prostatectomy for benignprostatic hyperplasia (BPH, N=2).

Tissue Preparation

Fresh tissue was obtained within 15 minutes of surgical removal.Approximately one gram of gross tumor was taken from a palpable tumornodule from 14 specimens. One gram of normal prostate tissue wasobtained from the prostatic lobe contralateral to the tumor nodule in 13specimens. One gram of BPH tissue was obtained from the periurethralregion of the contralateral lobe in 12 specimens and 25-30 grams fromeach of the 2 open prostatectomy specimens. All tissues removed werehistologically confirmed with hematoxylin and eosin sections on both theproximal and distal ends of the section.

Purification of Nuclear Matrix Proteins

Nuclear matrix proteins were isolated according to the methodology ofFey and Penman (Proc. Nail. Acad. Sci., USA, 85:121-125, 1988). Briefly,fresh human prostate tissue was minced into small (1 mm³) pieces andhomogenized with a Teflon pestle on ice with 0.5% Triton X-100 in asolution containing 2 mM vanadyl ribonucleoside (RNAase inhibitor)containing 1 mM phenylmethylsulfonyl fluoride (serine proteaseinhibitor) to release the lipids and soluble proteins. Extracts werethen filtered through a 350 micron nylon mesh and extracted with 0.25 Mammonium sulfate to release the soluble cytoskeletal elements. DNasetreatment at 25° C. was used to remove the soluble chromatin. Theremaining fraction contained intermediate filaments and nuclear matrixproteins. This fraction was then disassembled with 8 M urea, and theinsoluble components, which consist principally of carbohydrates andextracellular matrix components, were pelleted. The urea was dialyzedout and the intermediate filaments allowed to reassemble and wereremoved by centrifugation. The nuclear matrix proteins were then ethanolprecipitated. Protein concentrations were determined with theComassie^(R) Plus protein assay reagent kit (Pierce, Rockfcrd, Ill.)with bovine serum albumin as a standard. For preparation for gelelectrophoresis, the nuclear matrix proteins were redissolved in asample buffer consisting of 9 M urea, 65 mM 3- (cholamidopropyl)dimethylamino)-1-propanosulfonate, 2.2% ampholytes and 140 mMdithiothreitol.

Two-Dimensional Electrophoresis

High resolution two-dimensional gel electrophoresis was carried oututilizing the Investigator 2-D gel system (Milligan/Biosearch, Bedford,Mass.) (Patton, W. F., et al., BioTechniques, 8:518-527, 1990.One-dimensional isoelectric focusing was carried out for 18,000 V-husing 1 nm×18 on tube gels after 1.5 hours of prefocusing. The tube gelswere extruded and placed on top of 1-mm pre-cast 10% Tris-acetate sodiumdodecyl sulfate Duracryl™ (Millipore, Colo., Bedford, Mass.) hightensile strength (HTS) polyacrylamide electrophoresis slab gels and thegels were electrophoresed with 12° C. constant temperature regulationfor approximately 5 hours. Gels were fixed with 50% methanol and 10%acetic acid. After thorough rinsing and rehydration, gels were treatedwith 5% glutaraldehyde and 5 mM dithiothreitol after buffering with 50Mm phosphate (pH 72). Gels were stained with silver stain using themethodology of Wray (Wray, W. et al., Anal. Biochem, 118:197-203, 1981)(Accurate Chemical Co., Inc., Westbury, N.Y.). Fifty micrograms ofnuclear matrix protein were loaded for each gel. Protein molecularweight standards were determined with the GELCODE (Dacheng, H., et al.,J. Cell Biol., 110:569-580, 1990), protein molecular weight market kit(MW 12,400-97,400), (Pierce, Rockford, Ill.). Isoelectric points weredetermined using carbamylated creatine kinase standards (pH 7.0-4.950,(BDH Limited, England)). Only protein spots clearly and reproduciblyobserved or absent in all samples from the various tissues wereconsidered when determining variations in nuclear matrix proteinsbetween tissues.

There was marked similarity seen in the nuclear matrix protein patternsbetween patients with approximately 120 of 150 proteins spotsconsistently seen from patient to patient. Fourteen nuclear matrixproteins were identified that were consistently present or absent whencomparing normal prostate BPH and prostate cancer. A protein (PC-1),with a molecular weight of 56 Kd and pl=6.58, represented a nuclearmatrix protein that appeared in all (14/14) human prostate cancerspecimens studied but was not detected in any normal prostate (0/13) orBPH tissue (0/14).

FIG. 1 shows nuclear matrix protein composition of normal human prostate(A), benign prostatic hyperplasia (B) and prostate cancer (C). Fourproteins known to be present in most nuclear matrix preparations, laminA, lamin B, lamin C and actin were identified based upon previouslyreported molecular weights and isoelectric points (Fey, E. G., et a.,Proc. Natl. Acad. Sci. USA, 85:121-125, 1988) and labeled on FIG. 1A asLA, LB, LC and A respectively.

FIG. 1 demonstrates the typical high resolution two-dimensional gelelectrophoresis patterns for nuclear matrix proteins isolated fromnormal human prostate (FIG. 1A), human BPH (FIG. 1B) and human prostatecancer (FIG. 1C). Gel spots differing between normal prostate, BPH andprostate cancer (in all specimens examined) have been marked with arrowsand identified with labels corresponding to those in Table 1. Table 1demonstrates the molecular weight and isoelectric points of the 14different protein spots found to be consistently present or absent whencomparing nuclear matrix proteins from normal prostate, BPH and prostatecancer tissue for this group of 21 patients.

FIG. 2 summarizes the location of the protein spots that differedbetween the various tissues and shows specific nuclear matrix proteinsin BPH and prostate cancer. Schematic of major tissue specific nuclearmatrix proteins of normal prostate, BPH and prostate cancer.Abbreviations: kD--molecular weight in thousands, SDS-PAGE--sodiumdodecyl sulfate-polyacrylamide gel electrophoresis and pl--isoelectricpoint and BPH--benign prostatic hyperplasia.

No NMPs were detected which were present only in BPH and were absent innormal prostate and prostate cancer. Likewise, no NMP's were presentboth in normal prostate and prostate cancer, but were absent in BPH.PC-1 (molecular weight 56 Kd and isoelectric point 6.58) represents anNMP seen only in human prostate cancer tissue and was consistentlyabsent in all normal prostate and BPH samples. In additional testing,PC-1 was found in kidney and bladder cancer specimens, but was notdetected in normal kidney or bladder tissue.

The absence of NPB-1-7 and NP-1-3 in malignant cells suggests that genesencoding these NMPs may function as cancer suppressor genes. This isespecially true with respect to NP-1-3 which were also absent in benignhyperplastic tissue.

                  TABLE 1                                                         ______________________________________                                        NUCLEAR MATRIX PROTEINS FROM FRESH NORMAL                                     PROSTATE. BPH AND PROSTATE CANCER TISSUE                                                              Normal BPH     Cancer                                 Protein                                                                              M.sub.r  pl      (N = 13)                                                                             (N = 14)                                                                              (N = 14)                               ______________________________________                                        NPB-1  17,000   6.91    +      +       -                                      NPB-2  17,000   8.30    +      +       -                                      NPB-3  12,000   8.40    +      +       -                                      NPB-4  12,000   6.91    +      +       -                                      NPB-5  43,000   6.27    +      +       -                                      NPB-6  43,000   6.22    +      +       -                                      NPB-7  43,000   6.14    +      +       -                                      NP-1   12,000   7.50    +      -       -                                      NP-2   11,500   7.62    +      -       -                                      NP-3   11,000   8.30    +      -       -                                      BPC-1  42,500   5.80    -      +       +                                      BPC-2  42,000   5.73    -      +       +                                      BPC-3  41,000   5.64    -      +       +                                      PC-1   56,000   6.58    -      -       +                                      ______________________________________                                         NP--Normal Prostate                                                           BPH Benign Prostate Hyperplasia.                                              PC--Prostate Cancer; NPB--Both Normal and Benign Hyperplasia Prostate         Tissue; BPC--Benign Hyperplasia and Cancerous Prostate Tissue.           

The designation of each protein corresponds to the identified proteinsin FIG. 1.

EXAMPLE 2 Models for Progression from Normal Prostate Cells to ProstaticCancer

Although the precise molecular and/or environmental events necessary forthe development of prostatic disease are largely unknown, it has beenwell established that the development of prostate cancer is a multistepprocess (Carter, H. B., et al., J. UroL., 143:742-746, 1990).Epidemiologic studies based on the original work of Ashley (Ashley, D.F. B., J. Path. Bact., 90:217-225, 1965) and Armitage and Doll (ArmitageP. and Doll, R., Brit J. Cancer, 8:1-15, 1954) using age specificincidence rates for prostate cancer and BPH in the United Statesdemonstrate that development of BPH is most likely a two-step processwhile the development of clinically evident prostate cancer most likelyinvolves a multi-step (greater than 2 event) process (Carter, H. B., etal., J. Urol., 143:742-746, 1990).

Two different models can be postulated for the progression of a normalprostatic epithelial cell to either BPH or prostate cancer. FIG. 3 showstwo models of multistep progression from normal prostate (Normal) tobenign prostatic hyperplasia (BPH) or to prostate cancer (Cancer). ModelI predicts that similar events occur in both pathways. Model II predictsdifferent events occurring when progressing from normal to BPH as whenprogressing to cancer. The first model (Model I) predicts that the earlyevents for progression from either normal to BPH or normal to prostatecancer are similar (events A-B in Model I). The second model (Model II)predicts that progression for BPH and cancer would undergo differentevents (events A-B versus events E-H). Using the presence or absence ofnuclear matrix proteins as a phenotypic marker to test these models, itwould be predicted that Model I would be satisfied if a specific groupof protein spots were either absent or present in both BPH and prostatecancer (NP 1-3, BPC 1-3) and additional protein spots were present orabsent in only prostate cancer (NPB 1-7 or PC-1). Thus all of thedifferences observed in the nuclear matrix proteins satisfied Model I.In order to satisfy Model II, a protein(s) need be present or absent inBPH only and this was not observed in any samples. Thus, these datasupport Model I in which similar phenotypic expressions are occurring inthe nuclear matrix of cells progressing to BPH as those cellsprogressing to prostate cancer. As a result, the NMPs of the inventioncan be used to monitor and detect the stage and progression of a cellproliferative disorder, such as prostate cancer, from normalcy to benigndisease to malignancy. This information, in turn, can be used toinitiate appropriate therapy.

The foregoing is meant to illustrate, but not to limit, the scope of theinvention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

We claim:
 1. An in vitro method for detecting a prostatic or urogenitalcell proliferative disorder in a subject comprising contacting aspecimen suspected of containing a protein selected from the groupconsisting of Normal and Benign Hyperplasia Prostate Tissue-1 (NPB-1),Normal and Benign Hyperplasia Prostate Tissue-2 (NPB-2), Normal andBenign Hyperplasia Prostate Tissue-3 (NPB-3), Normal and BenignHyperplasia Prostate Tissue-4 (NPB-4), Normal and Benign HyperplasiaProstate Tissue-5 (NPB-5), Normal and Benign Hyperplasia ProstateTissue-6 (NPB-6), Normal and Benign Hyperplasia Prostate Tissue-7(NPB-7), Benign Hyperplasia and Cancerous Prostate Tissue-1 (BPC-1),Benign Hyperplasia and Cancerous Prostate Tissue-2 (BPC-2), BenignHyperplasia and Cancerous Prostate Tissue-3 (BPC-3), and ProstateCancer-1 with a reagent which binds to at least one of the protein anddetecting binding of the reagent to at least one of the proteins in thespecimen, wherein the presence of at least one of the proteins isindicative of a prostatic or urogenital cell proliferative disorder. 2.The method of claim 1, wherein the reagent is an antibody.
 3. The methodof claim 2, wherein the antibody is polyclonal.